Modeling of three-dimensional innervated epidermal like-layer in a microfluidic chip-based coculture system

Reconstruction of skin equivalents with physiologically relevant cellular and matrix architecture is indispensable for basic research and industrial applications. As skin-nerve crosstalk is increasingly recognized as a major element of skin physiological pathology, the development of reliable in vitro models to evaluate the selective communication between epidermal keratinocytes and sensory neurons is being demanded. In this study, we present a three-dimensional innervated epidermal keratinocyte layer as a sensory neuron-epidermal keratinocyte co-culture model on a microfluidic chip using the slope-based air-liquid interfacing culture and spatial compartmentalization. Our co-culture model recapitulates a more organized basal-suprabasal stratification, enhanced barrier function, and physiologically relevant anatomical innervation and demonstrated the feasibility of in situ imaging and functional analysis in a cell-type-specific manner, thereby improving the structural and functional limitations of previous coculture models. This system has the potential as an improved surrogate model and platform for biomedical and pharmaceutical research.

The text doesn't follow, if 2D works why is 3D needed? "disease skin models to guide therapeutic, pharmaceutical, and industrial discoveries." is very generalized and cannot be applied to this study in this way. More concrete examples are needed. Since the hydrogel lacks any dermis derived cell type, even the fibroblast, the hydrogel cannot be refered to as a demis in this model. What is the sourse of the SNs? "complicated basal and spinous layer of epidermis" why complicated, what about granular layer and stratum corneum. Static models have these, microfluidics not needed. The term "airway" culture suggests respiratory not skin.
Results: Fig 1 does not show any results. Terminology in this entire section needs significantly revising: Reconstitution of epidermis on ECM hydrogel. What is the idea between the flat and sloped culture method. The reason for air exposure is clear as literature from many studies over many years shows that this supports epidermal differentiation Why do you need medium flow and a chip. Why not use a simple static transwell system rather than the chip? Please supply heamatoxylin & eosin histology of the epidermis in order to see laters as indicated in fig 1. Dapi staining and the weak K10 staining are not enough to support the conclusions. Also supply K5 and Ki67 in order to see the quality of the basal layer and loricrin to see quality of the granular layer. I miss the flat air exposed condition, this would also stimulate stratification of keratinocytes. The word "integrity" is misplaced Fig 4: what is A1 A2 and A3. No significance is shown in fig 4 g and h indicating no effect of tested sustances.
Discussion: Why is the microfluidic chip needed. Would the same results be obtained in an easier more scalable transwell system. If so, what effect would this have on the novelty presented here when compared to the studies presented in Supplementary table 1? I miss the static air exposed model and detailed (immuno)histology of the epidermis. The model does not contain a dermis equivalent as no cells are present in the hydrogel. Many studies ranging back 30 years describe static 3D reconstructed human epidermis on fibroblast populated collagen hydrogels. What would happen if you cultured SN cells underneath such a construct. Has a similar study already been done?
Supplementary images are of very poor quality. S4d shows only a monolayer of keratinocytes which is not even intact (Dapi) and very little K10. These images do not support the conclusion of epidermal stratification as suggested in S4c. S3 and S5 black / white image extremely poor quality to draw results from Table S1 is unclear. Eg what is yellow block. What different cell types are in the different skin models, legend is unclear

Reviewer: 1
The manuscript by Ohk et al. describes an in vitro "skin-nerve" model based on co-cultures of human keratinocytes and rat dorsal root ganglion (DRG) sensory neurons in microfluidic devices in a three-dimensional configuration. The DRG cells and the keratinocytes are separated by two layers of collagen hydrogels in a configuration that allows the neurons to extend their axons across the collagen layer to reach the keratinocyte compartment. The keratinocytes have been differentiated through air exposure to mimic epidermis formation in the "skin" compartment. The authors show that the DRG neurites can extend into the differentiated "epidermis" and release CGRP in response to stimulation of the keratinocyte compartment with capsaicin or phorbol ester. The authors conclude that the co-culture system described can be used for disease modelling and toxicity testing.
From a technical point of view, the methodology described in the paper is very interesting. However, my major concern is that a more in-depth characterisation of the model would be required to support the conclusions drawn in the paper, particularly in relation to the utility of the described co-culture platform for disease modeling.
1. It would be important to know the extent of the fluidic isolation (i.e. permeability of the hydrogel layers) between the DRG and keratinocyte compartments in the PDMS devices. This issue has not been addressed in the manuscript. Can substances such as CGRP or capsaicin permeate through the collagen hydrogels between the compartments? (Answer) We deeply appreciate the reviewer's comment. Fluidic isolation by the skin layer is important to trust the signal transduction from keratinocytes to DRGs. As the reviewer pointed, the manuscript should prove barrier function of the skin layer for the smallest molecule involved in the experiments. The molecular weight of CGRP is known to about 3.789 kDa (human) and that of Capsaicin is about 33.5 kDa. The authors conducted permeability test using FITCconjugated dextran, which has a molecular weight of 3.984 kDa. In brief, 90 μl of serum-free media was first filled in both channels. Under the fluorescent microscope, 10 μl of 250 μM FITC-dextran solution was added to the HEK channel and 10 μl of serum-free media was to SN channel at the same time. Diffusion of the FITC-dextran was monitored by time-lapse images. The FITC-dextran signal was successfully blocked by the intact keratinocyte barrier of three devices for 2 hours, not diffusing into the ECM hydrogel. 2. To demonstrate the 3D outgrowth of the neurites into the collagen hydrogels, a set of 3D Zstack projections or 3D reconstruction of the fasciculated neurites in the collagen hydrogels would be beneficial. The current images do not give any indication of the 3D outgrowth of the neurites into the hydrogel layers.
(Answer) Thank you for the suggestion. We added 3D images of three-dimensionally reconstructed neurites (stained by Tuj-1 and NF-M) near the HEK layer (stained by K10 and K14) (Fig. 1d). They show the 3D outgrowth of the neurites into the hydrogel toward the HEK layer.  3. It is not clear if non-peptidergic sensory neurites can also enter the hydrogel layers and innervate the keratinocyte compartment. Given the functional heterogeneity of DRG sensory neurons, it would be of interest to characterise the neurites crossing into the keratinocyte compartment using markers including NF200, IB4, CGRP and TRPV1 to ascertain representation of different neuronal cell types in the co-cultures.
(Answer) We appreciate the helpful comments and suggestions. To identify the types of the sensory neurites, we immunostained DRGs by NF200 (against myelinated (Aβ, Aδ) fibers, TRPV1 (against small diameter C fibers), IB4 (against non-peptidergic C fibers) and CGRP (against peptidergic C fibers) [1,2]), as the reviewer commented. The images were added as fig.  4e and supplementary fig. 8a. In the ECM hydrogel far from HEK layer (A1 in Fig. 4b), large number of TRPV1 neurites and double positive neurite by TRPV1 and NF200 were found. However near the HEK layer (A2 in Fig. 4b), only TRPV1 neurites were found, without NF200 expressing ones. In ECM hydrogel near HEK layer, neurites expressing IB4 or CGRP were found. However only CGRP positive neurites projected inside the HEK layer ( Fig. 4e and Supplementary Fig. 8a).
Various subtypes of 3D sensory neurons in ECM hydrogel were found in the developed coculture platform, as summarized; 1) far from HEK layer : myelinated C-fibers (TRPV1 and NF200) 2) near HEK layer : peptidergic or non-peptidergic C-fibers (CGRP or IB4 with TRPV1) 3) Inside HEK layer : only peptidergic C fibers (CGRP) 4. Figure 4 shows a significant increase in the basal levels of CGRP release in the co-cultures compared to single cultures of sensory neurons (figures 4f and supplementary 5b). However, the capsaicin-evoked CGRP release appears to be higher in sensory neuron cultures alone (ratios given in Figure 4f). The authors concluded that "the integrity of the innervating sensory neurites is preserved" in the co-cultures, however, this could suggest a reduced sensitivity to capsaicin or decreased CGRP content in DRG neurons maintained in the co-cultures.
(Answer) We certainly agree with the reviewer and appreciate the comments. The graphs of the previous manuscript were vague, misleading readers. We performed additional experiments and revised manuscript and figures, to draw clearer conclusion than before.
1) The basal level of CGRP in HEK-SN co-culture was approximately the summation of CGRP in HEK mono-culture and in SN mono-culture (Fig. 5d, left). The developed platform seemed to preserve the CGRP contents in HEK-SN co-culture. 2) Capsaicin and 4α-PDD applied on the HEK layer could not permeate through the HEK layer, as proved in Fig. 3f-g. 3) CGRP contents measured in the medium collected from the SN channel did not correlate to the concentration of the applied stimulus (capsaicin or 4α-PDD) on the HEK layer. However when the stimulus applied to the HEK layer with SN, the measured concentrations were proportional to the concentrations of the applied stimulus on the HEK layer (Fig. 5d, center and right, supplementary fig. 5) 4) Due to the increase of the basal level of CGRP in HEK-SN co-culture, the absolute CGRP levels in HEK-SN co-culture cases were much higher than those in HEK mono-culture cases (marked by ### in Fig. 5d). 5) CGRP contents in SN mono-culture cases were unstable, due to the reduced viability of SNs by the stimulus. Without HEK layer, neurons quickly loose their morphology by the directly applied capsaicin and 4α-PDD, while the neurons in co-culture still maintained their network in one day after the 4α-PDD treatment (Supplementary fig. 11).
In conclusion, the applied stimulator on the HEK layer did not directly reach the neurons. Without SN, the applied stimulator did not affect the CGRP concentration. The integrity of HEK-SN can be verified by 1) the role of mediators (i.e. ATP) from keratinocytes activating surrounding DRG neurons, and 2) TRPV channels between the DRG neurites innervating HEK layer and neighboring keratinocytes [3,4].  concentration without stimulation in mono-and co-cultured groups. Error bars indicate standard deviation (*p < 0.05, **p < 0.01, ***p < 0.001; n = 4 (a, left), n = 7 (a, right) and n = 4 (b) for each case). Figure 11. HEK layer protected sensory neurons from 4α-PDD treatment. (a) Phasecontrast images of HEK, SN mono-and HEK-SN co-cultures after 4α-PDD treatment. Sensory neurons(SNs) without HEK layer were shrunk after 0.1, 0.2 mM 4α-PDD treatment but not in the co-culture condition. (b) The magnified image of SNs (red box in (a)) The scale bars represent 250 μm.

Supplementary
5. Statistical analysis of the CGRP release ratios is not discussed in the manuscript. The pvalues for statistically non-significant data should also be provided. It would be more informative to present the actual capsaicin or 4alphaPDD evoked CGRP release levels for comparison.\ 6. It is not clear if the CGRP release ratios are not significantly different, or that no statistical analysis of the ratios has been carried out. 7. The authors conclude that "the integrity of the innervating sensory neurites is preserved" in the co-cultures, however, whether these neurites are capable of generating and conducting action potentials to their somas, has not been addressed. Characterisation of the electrophysiological properties of the neurites in the co-cultures, using calcium imaging, for instance, would be of interest here.
(Answer) We deeply appreciate the reviewer's suggestions and performed additional experiments to acquire calcium imaging signal in the sensory neurites in HEK-SN co-culture condition. We added fig. 5b and supplementary fig. 10.
Before capsaicin treatment, we observed spontaneous calcium flux on individual DRG neurons but could not find calcium expressing DRG neurites. However 3 seconds after treating 0.1 mM capsaicin on the HEK layer, capsaicin-evoked calcium flux was observed at the tiny DRG neurites near the HEK layer ( Fig. 5b & supplementary fig. 10). The significant increase was quantitatively confirmed by normalized fluorescent intensity.  8. The authors conclude based on the morphology of the neurites immunostained with PGP9.5 antibody that the neurites entering the epidermis layer "…form cutaneous nerves [endings]". However, it is not clear if these neurites terminate once entering the differentiated keratinocyte layer or continue to extend growth cones in the "epidermis" layer. It would be important to determine if the neurites projecting through the differentiated keratinocyte layer terminate, in a similar manner to the projections in vivo, or if they continue to grow via growth cones. For instance, immunolabeling with growth cone markers such as gap-43 could be informative.
(Answer) The reviewer was certainly right. We agreed with the suggestion and immune-stained the DRG neurites entering the epidermis layer with gap-43 (against growth cone) and TRPV1 (against free nerve ending) antibodies [5,6]. Supplementary fig. 8 was added.
DRG neurons migrating into the ECM hydrogel expressed gap-43 and TRPV1. Neurites entered in HEK layer also expressed both gap-45 and TRPV1, confirming extending growth cone and formation of free nerve ending in the epidermis layer. Interestingly some neurites only expressed TRPV1 (white arrowheads). After invading HEK layer, DRG neurites seemed to lose growing capability. (c) DRGs expressed TRPV1 on their soma and neurites and many of the neurites also expressed gap-43, the growth cone marker. In the HEK layer, some of neurites only expressed TRPV1, which is the marker of free nerve ending. The scale bars represent … 100 μm in (c, top) and 25μm in (c, bottom). 9. Figure 3 and supplementary figure 4d show the formation of the spinous layer using K10 immunostaining. Given the importance of the basal layer of the skin, it would be interesting to characterise the undifferentiated keratinocyte layer using K5 immunostaining.
(Answer) The reviewer was certainly right. We performed additional experiments for keratins by K5 / K14 (in basal) and K10 (differentiating suprabasal), and for terminally differentiated epidermal cells by Loricrin [7,8]. Fig. 4f and two supplementary figures (7b and 8b) were added. Fig.4f show that the alignment of K10 and K14 expressing cells is much stable in HEK-SN coculture case, K10 expressing cells above the K14 cells. When cultured with DRG neurons, polarity of the co-cultured HEK layer became stable, by reduced invasion into the ECM hydrogel and enhanced proliferation in the top layer (Supplementary fig.9). Consistent K14 and K10 alignment was noted in HEK layer co-cultured with DRG SNs (Supplementary figure 8b).  10. TRPV4 has been shown to be present on sensory nerve endings and keratinocytes in the skin. Figure 4h suggests that there is an increase in the 4alphaPDD mediated release of CGRP from co-cultures of SN and keratinocytes compared to SN alone. However, the data for keratinocyte cultures alone are not presented. This would be of interest since the presence and release of CGRP from human keratinocytes has been reported (e.g Pain. 2011 Sep;152(9):2036-51). If CGRP is released from Keratinocytes in response to stimulation by 4alphaPDD, this would explain the additional CGRP release seen in the co-cultures compared to single SN cultures.  HEKs and DRG sensory neurons were doubleimmunostained with each TRPV1 antibody (TRPV1-DRG(D) and TRPV1-HEK(H)) ... (d) CGRP concentration measured by ELISA in the medium collected from SN channel. Cases include HEK or SN mono-culture and HEK-SN co-culture; CGRP concentration without stimulation (left), the ratio of the CGRP concentration under capsaicin stimulation (middle), and the ratio of CGRP concentration under 4α-PDD stimulation (right). Error bars indicate standard deviation (*,#p < 0.05, **,##p < 0.01, ***,###p < 0.001; * for the ratio of CGRP concentration and # for the actual values of CGRP concentration; n = 11 (left), n = 4 (middle) and n = 7 (right)).
11. The advantages of the 3D model described in this manuscript over other reported co-culture or microfluidic platforms (e.g. Biomed Microdevices (2017) 19: 22, Biomaterials (2017)116:48 and PLOS ONE 8 (11), e80722) are unclear. Ultimately a proof of concept experiment to demonstrate the utility of the co-culture system in disease modelling would be of interest.
(Answer) The reviewer is certainly right. In this manuscript, the authors focused more on the realization of 3D reconstitution of cutaneous nerve in microfluidic device. Challenges and achievement of this manuscript in terms of model development are listed in supplementary table 1, carefully revised and updated during revision. Syndromes on cutaneous nerves have complicated disease mechanism, with list of drugs and molecules. Complexity exists with unique delivery routes, stromal cells and ECMs, which should be carefully verified in the future study.
12. Furthermore, the disadvantages of the model should be clearly addressed in the discussion, particularly in the view of the inter-species nature of this model which could significantly limit its utility for human disease modeling and toxicity testing.
(Answer) We appreciated the reviewer's comment. Discussion was revised.

Discussion
... There are several challenges of the developed microfluidic platform. First, maturity of the neurons in the epidermis-mimicking layer has not yet been fully confirmed. A previous study showed that a subset of IB4-positive non-peptidergic C-fibers terminate exclusively in the stratum granulosum of the epidermis 46 , which was not clearly identified in our platform ( Fig.  S7-c). Only weak projections of IB4-positive non-peptidergic fibers were observed ( Fig. 4e and Fig. S8-a) and free nerve endings of the peptidergic c-fibers seemed to not be fully mature, coexpressing gap-43 and TRPV1. It is known that most CGRP-positive fibers terminate in the stratum spinosum (spinous layer) 46 , and the maturation of the spinous layer is required to mature peptidergic c-fiber-free nerve endings. The interspecies combination of HEKs and DRG SNs is another challenge. Utilization of human induced pluripotent stem cell (hiPSC)derived sensory neurons 59 and Schwann cells with primary keratinocytes could address this issue, with the additional potential benefit of the development of patient-specific models.
The study describes co-culture of epidermal keratinocytes and sensory neurons in a microfluidics device and cultured at the air liquid interface to enable keratinocyte differentiation and stratification. Neuropeptide secretion by SNs and neurite migration is reported e.g. calcitonin gene-related polypeptide. The authors claim that their "observations validated the potential of the proposed model as an in vitro cutaneous nerve model for disease studies and drug toxicity tests". This is not shown in the study. No disease model and limited drug testing is performed, with no statistical significance. Therefore this claim is exaggerated. Although interesting, it remains a potential model which requires much further extensive investigation before publication in Nature Communications. Furthermore, use of English language needs extensive correcting throughout.
(Answer) We appreciate the reviewer's sincere and helpful comments. The whole manuscript was carefully revised by the authors with additional experiments. After revision, language was also checked by editing service with attached certification of English editing.
Title: is misleading as the study describes only one type of skin cell, the keratinocyte. Cutaneous indicated the complexity of the skin organ. This terminology needs correcting throughout.
(Answer) We certainly agree with the reviewer. Skin includes multiple layers of epidermis and dermis, containing vascular network and nervous systems. Epidermis also bears heterogeneity, by multiple aspects of keratinocytes from non-differentiated to fully differentiated. This

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manuscript deals with complicated aspects of epidermis and nervous systems with sensing capabilities. Revised manuscript includes immunofluorescent images of various phases of keratinocytes and DRG neurons, taking the initial step of 'cutaneous' nerve on a chip. We revised large part of the manuscript but hope to keep the word of 'cutaneous nerve on a chip' in title to encourage recent progress of 'tissue on a chip' field.
Abstract: needs revising according to general comment (Answer) Not only the abstract, the whole manuscript was carefully revised.

Abstract
There is a growing need for in vitro models of skin that help accelerate the development of drugs and replacement of animal experiments. This study describes the development of a three-dimensional epidermal nerve model in the extracellular matrix (ECM) incorporating a microfluidic chip. The proposed model co-cultured human epidermal keratinocytes (HEKs) and sensory neurons (SNs) across the ECM hydrogel. We investigated 3D neurite outgrowth into ECM hydrogel toward the epidermis-mimicking layer cultured under an airliquid interface. The model revealed the direct integration of the epidermis and innervation with tiny neurites of SNs by the calcitonin gene-related polypeptide, and demonstrated the evoked calcium flux on tiny innervating neurites following external stimulation of the epidermis. These observations validate the potential of the proposed model as an in vitro epidermal nerve model for investigating cutaneous sensory innervation.

Introduction
Experimental approaches for modeling the structure of skin in vitro have been studied since the 1980s 16,17 . Profound restrictions in animal experimentation have driven the development of in vitro skin models that simulate actual skin to test topical or systemic drug actions in the fields of pharmaceutics and cosmetics 18 .
5. 3D Skin equivalent models have existed for 30 years without microfluidics. Porous films are generally known as transwell inserts. The text doesn't follow, if 2D works why is 3D needed?
(Answer) The authors agreed with the reviewer's concern, and carefully arranged the introduction with more details. As the reviewer commented, 3D skin models have formed multiple layers of epidermis in transwell inserts. However microfluidic culture is beneficial by the precise 3D reconstitution with epidermis layer on the collagen rich dermal-mimicking hydrogel. 3D spatial alignment in microfluidic platform enabled co-culture of 3D DRG sensory neurons, showing peptidergic interaction with epidermis mimicking layer and exhibiting various somatosensory neuronal subtypes at the same time. 3D microfluidic platform enabled the process of this epidermal innervation and clear visualization of the reconstituted units' functions.
In the revised introduction, previous references were carefully reviewed and re-arranged to present the benefit of the developed microfluidic skin platform.

(revised manuscript) Introduction
Experimental approaches for modeling the structure of skin in vitro have been studied since the 1980s 16,17 . Profound restrictions in animal experimentation have driven the development of in vitro skin models that simulate actual skin to test topical or systemic drug actions in the fields of pharmaceutics and cosmetics 18 . As human skin equivalent (HSE) models have been developed in transwells, it has become possible to study the function of the skin including its barrier properties by mimicking the epidermal layer 64 . However, the complicated structure of skin, with its various appendages, has been a major challenge to the implementation of complicated and accurate skin models. Some pioneering studies have cultured human epidermal keratinocytes (HEKs) and dorsal root ganglia (DRG) sensory neurons (SNs) from various species (e.g., rat, porcine, and mouse) together 60, 62, 63 , and showed in vitro interactions between HEKs and DRG SNs by APT-mediated signal transfer from keratinocytes to DRG SNs 10,61,63 . An increase in the growth of keratinocytes by neuropeptide signaling from DRG SNs 65 or elevated intracellular calcium in DRG SNs by stimulation of the epidermal layer has also been presented 66 . These studies have enhanced the complexity of the in vitro skin model, but only by co-culturing without direct contact or by projected DRG neurites onto the HEK layers. The studies could not replicate the complexity of innervating sensory nerves in epidermal layers. Recently reported microfluidic skin models have been successful in imitating major functions of the skin layer, particularly in blocking or transporting specific molecules 19,20,21 . These models have provided benefit through the temporal application of drugs and also through the precise regulation of fluidic culture conditions, but fail to replicate the 3D reconstitution of innervating sensory nerves in epidermal layer, the achievement of which would improve our understanding of the mechanisms underlying skin sensation.
6. "disease skin models to guide therapeutic, pharmaceutical, and industrial discoveries." is very generalized and cannot be applied to this study in this way. More concrete examples are needed.
(Answer) We appreciate the reviewer's comment. We investigated the reconstruction of peptidergic sensory nerve endings and revealed the functional interactions between epidermal keratinocytes and DRG neurons in the developed microfluidic platform. With this platform, diseases can be modeled, i.e. hyper-innervation, the typical symptom in the skin of Atopic dermatitis patients by imbalance between nerve elongation and repulsion factors [9].
In this manuscript, we focused more on the realization of 3D reconstitution of cutaneous nerve in microfluidic platform. Challenges and achievement of this manuscript in terms of model development are listed in supplementary table 1, carefully revised and updated during revision. Syndromes on cutaneous nerves have complicated disease mechanism, with list of drugs and molecules. Complexity exists with unique delivery routes, stromal cells and ECMs, which should be carefully verified in the future study. In revision, we deleted the sentence.
7. Since the hydrogel lacks any dermis derived cell type, even the fibroblast, the hydrogel cannot be refered to as a demis in this model.

(Answer)
We substitute the word 'dermis' with 'dermis-mimicking' in the whole manuscript. Thank you for the comment.
8. What is the sourse of the SNs?
(Answer) We already described the source of SNs in Methods, but changed the word SNs into DRG SNs in revision. Apologies for the confusion.

Primary DRG isolation
Primary DRG were isolated from embryonic day 15 rat embryos (KOATECH, Gyeonggi, South Korea) via a surgical procedure… 9. "complicated basal and spinous layer of epidermis" why complicated, what about granular layer and stratum corneum. Static models have these, microfluidics not needed.
(Answer) Thank you for the comment. We performed additional experiments for keratins by K5 / K14 (in basal) and K10 (differentiating suprabasal), and for terminally differentiated epidermal cells by Loricrin [7,8].  10. The term "airway" culture suggests respiratory not skin.
(Answer) We appreciate the comment and substituted 'airway culture condition' with 'air-liquid interface culture condition'. Definition of 'air-liquid interface culture condition' in our model was also added in revision.  12. What is the idea between the flat and sloped culture method. The reason for air exposure is clear as literature from many studies over many years shows that this supports epidermal differentiation.
(Answer) Thick epidermal layer prohibited leakage of medium into the air channel, and maintained air-liquid interface in the microfluidic platform both under flat and sloped conditions. As the reviewer commented, air exposure of HEK layer in transwell supports epidermal differentiation. However in transwell, heavy soluble components in medium below the HEK layer could not diffuse toward HEK layer against gravity. The sloped microfluidic model precisely controls tiny flow from SN channel toward HEK layer by the head difference between the HEK channel reservoirs and SN channel reservoirs. It enhanced communication between SNs and HEK layer, which is advantageous against transwell model.

Discussion
Although the K14 and K10 were expressed in the HEK layer, the loricrin expression was not separated with other layers and Ki67 was rarely expressed in this condition (Fig S7-b and S7c).
15. I miss the flat air exposed condition, this would also stimulate stratification of keratinocytes.
(Answer) We performed additional experiments to check the role of the flat air-liquid interface condition to form epidermis-mimicking layer. We immunostained epidermis-mimicking layers cultured under sloped or flat (non sloped) air-liquid interface conditions. The K10 expression was reduced in HEK layer under the flat (non sloped) air-liquid interface conditions than under sloped one. (Supplementary figure 7). Air-liquid interface seemed to be affected by pressure head generated by the head difference between the reservoirs (60 ul medium in the SN channel reservoirs and no medium in the HEK channel reservoirs).

Results
We found that applying a slope of 30° to the device was successful in assisting keratinocyte differentiation into a spinous layer 28,29 . However, the invasion of HEKs into the ECM hydrogel increased after day 4-6 in the sloped air-liquid interface condition ( Fig. S7-a). The removal of the slopes (non sloped air-liquid interface condition) reduced the invasion but also the expression of K10 (Fig. S7-a and S7-b) at the same time. Barrier functions of these constructed epidermis-mimicking layers were evaluated by permeability tests with 3.984 kDa FITC-conjugated dextran. The applied dextran in the HEK channel was fully blocked by the intact HEK layer for 2 hours (Figs. 3f and 3g). However, if there were defects in the HEK layer, the dextran quickly diffused into the ECM hydrogels (Fig. S6). 16. The word "integrity" is misplaced

Results
Without stimulation, CGRP was rarely detected in the medium of the monocultured HEK layer. Significantly increased amount of CGRP was detected in the medium of mono-cultured DRG SNs and even more in the medium of the co-cultured DRG SNs (Fig. 5d). This indicated the integrity of the cutaneous nerve bundles   17. Fig 4: what is A1 A2 and A3. No significance is shown in fig 4 g and h indicating no effect of tested sustances.
(Answer) The locations of A1, A2 and A3 were described in the figures and manuscript. We apologize for the confusion our figures caused.
To confirm the significance, we performed additional experiments and revised manuscript and figures.
1) The basal level of CGRP in HEK-SN co-culture was approximately the summation of CGRP in HEK mono-culture and in SN mono-culture (Fig. 5d, left). The developed platform seemed to preserve the CGRP contents in HEK-SN co-culture.
2) Capsaicin and 4α-PDD applied on the HEK layer could not permeate through the HEK layer, as proved in Fig. 3f-g. 3) CGRP contents measured in the medium collected from the SN channel did not correlate to the concentration of the applied stimulus (capsaicin or 4α-PDD) on the HEK layer. However when the stimulus applied to the HEK layer with SN, the measured concentrations were proportional to the concentrations of the applied stimulus on the HEK layer (Fig. 5d, center and  right, supplementary fig. 5) 4) Due to the increase of the basal level of CGRP in HEK-SN co-culture, the absolute CGRP levels in HEK-SN co-culture cases were much higher than those in HEK mono-culture cases (marked by ### in Fig. 5d). 5) CGRP contents in SN mono-culture cases were unstable, due to the reduced viability of SNs by the stimulus. Without HEK layer, neurons quickly loose their morphology by the directly applied capsaicin and 4α-PDD, while the neurons in co-culture still maintained their network in one day after the 4α-PDD treatment (Supplementary fig. 11).
In conclusion, the applied stimulator on the HEK layer did not directly reach the neurons. Without SN, the applied stimulator did not affect the CGRP concentration. The integrity of HEK-SN can be verified by 1) the role of mediators (i.e. ATP) from keratinocytes activating surrounding DRG neurons, and 2) TRPV channels between the DRG neurites innervating HEK layer and neighboring keratinocytes [3,4]. Presence of TRPV1 (in newly added fig. 5a) and TRPV4 ( fig. 5a) in HEK layer was proved, however capsaicin and 4α-PDD on mono-cultured HEK layer did not increase CGRP release ( fig. 5d). The external stimuli in our experiments only affected DRG neurons in HEK-SN co-culture condition.

Discussion:
18. Why is the microfluidic chip needed. Would the same results be obtained in an easier more scalable transwell system. If so, what effect would this have on the novelty presented here when compared to the studies presented in Supplementary table 1?
(Answer) As commented the introduction part, microfluidic skin culture enables cross-sectional investigation of 3D alignment of HEK layers and DRG SNs. 3D Growth of bundled DRG SNs into the dermis-mimicking hydrogel was sequentially monitored. The bundles were untangled when approaching near the HEK layers, and tiny neurites from the bundles invaded into the HEK layer. Neurite projection and distribution in the epidermis-mimicking layer could also be visualized. Capability of the clear visualization of 3D cell-cell and cell-ECM interactions is the most important advantage of microfluidic format. It is also well known that the secreted factors from cells are enriched in microfluidic scale. Microfluidic co-culture dramatically increases efficiency of paracrine cell-cell, which helps the emergence of new features not shown in transwell platform. Supplementary table 1 showed that more markers were found in the 3D microfluidic models, possibly due to the enhanced communication between cells by enriched components. Compared to the previous 3D co-culture models, our work successfully explored enhanced integrity of HEK layer and DRG neurons as shown in Supplementary table 1.

Discussion
A challenge is the optimization of the ECM hydrogel components in a microfluidic chip to facilitate the induction of both the active outgrowth of neurites and the well-organized epidermis-mimicking layer. The DRG SNs three-dimensionally growing into the ECM (type 1 collagen) hydrogel preferred a soft ECM hydrogel with a low concentration (COL1.5, 1.5 mg/mL) during attachment, which generated longer neurites as observed in previous studies involving DRG explants 35,36 . The laminin component in the ECM hydrogel hardly affected the growth of neurites as expected 37 , but did help the neurites to be arranged vertically toward the other side of the hydrogel. On the other hand, the HEKs required a stiff type 1 collagen ECM hydrogel to form a stable and thick layer. The microfluidic chip was designed to form a Janus ECM hydrogel with one side stiff (for HEKs) and the other side soft with laminin (for SNs). The HEKs formed a stable layer on the stiff side and reconstructed terminal differentiation in sloped air-liquid interface condition 29,38,39 .
... The 3D process of epidermal nerve formation involving the untangling of nerve bundles near the epidermis and the penetration of tiny neurites into the epidermis was newly discovered in the developed microfluidic platform. NF200-positive neurites terminated before the epidermismimicking layer, and the TRPV1-expressing neurites penetrated into the epidermis-mimicking layer. Aδ-fibers were known to be terminated in the dermis and the peptidergic and nonpeptidergic C-fibers in different epidermal layers 7 . The neurite distribution in the developed platform was similar to that previously reported in in vivo studies 46 .
... The integrity of the epidermal sensory nerves was further verified through calcium imaging. Calcium flux was observed to appear in the tiny neurites just below the epidermis-mimicking layer when the capsaicin was treated on the other side. Microfluidic platform is beneficial by the enhanced visualization of the signal transfer, by compartmented layers of multiple tissue types in the xy-plane 24 .

(revised table)
Supplementary Table 1   Table S1. 19. I miss the static air exposed model and detailed (immuno)histology of the epidermis. (Answer) As the reviewer commented, cellular dermal components (i.e. fibroblast and endothelial cells) are expected to affect proliferation, differentiation and maturation of epidermismimicking layer and DRG sensory neurons. However in microfluidic platform, stable culture of the cellular dermal components in dermis-mimicking hydrogel is very tricky, due to the active remodeling of hydrogel by the cells. The authors hope to explore it in future study.

Supplementary Information:
21. S4d shows only a monolayer of keratinocytes which is not even intact (Dapi) and very little K10. These images do not support the conclusion of epidermal stratification as suggested in S4c.
(Answer) We revised the immunostained images of K10 to support the epidermal stratifications in our epidermis-mimicking layer (Fig. 1d & Fig. 4f). Additional images described above would also be helpful for the confirmation. Thank you for the valuable comment.

(revised table)
Supplementary Table 1   Table S1. Previous in vitro models of sensory-skin interaction. 2D models presented the interaction of epidermal keratinocytes and peripheral DRG sensory neurons (DRG SNs), however not with epidermal layer of differentiated and basal keratinocyte layers. Human skin equivalent (HSE) models have in vivo like epidermal layer under air-liquid interface culture condition, but lack the direct and sequential visualization of epidermal free nerve ending formation in 3D. F-11: F-11 cell line (a mouse N18TG2 neuroblastoma X rat DRG sensory neuron hybrid cell line), H: human, P: porcine, R: rat, M: mouse, Cap: capsaicin, 4α-PDD: 4α-phorbol 12,13-didecanoate, ATP: adenosine triphosphate, Mech: mechanical stimulation, Elec: electrical stimulation, Heat: Heat stimulation.

Introduction and Results
Although the introduction is now better balanced, it does primarily focus on the sensory neurons and the interaction with the epidermal layers. An important aspect of the study according to the authors is the anatomy of the recapitulated epidermal-like cell layers. The introduction should provide a brief overview of the structure of the epidermis. Note that sensory neurons are pseudounipolar neurons and do not have dendrites. The reference to dendrites is confusing. All references throughout the manuscript to "sensory nerves" must be replaced with the appropriate terminology. In culture, it would generally be sensory axons or neurites.
The references to "Adelta" and "C-fibers" in cultures should be avoided, or carefully explained. For instance, the authors do not provide evidence that the TRPV1+ axons that have crossed to the epidermal-like layers are of Adelta type. These designations as Adelta or C-fibers are relevant to the in vivo anatomy and best avoided when describing cell culture models.
In several places, the authors refer to the "protective effects" of the epidermal-like layer on the sensory neurons/axons. However, it is not at all clear how the layer has a protective effect on sensory neurons in culture. This should be clearly explained and backed up by evidence. The manuscript has been extensively revised and improved English needs editing. In the results section the quality of the epidermis is overstated and needs to be down-played a bit. Having said this, it is exceptional to be able to do so many readouts and to image whilst the culture is in the chip thus enabling cell cell interactions to be investigated without disrupting the the tissue by having to remove it the chip first. The neuronal-epidermal interactions are very impressive.
Introduction: supplimentary table 1 is unclear as not all abbreviations and colours are explained. Importantly it is incomplete as many 3D skin models show e.g. loricrin (in stratum granulosum) and many authors have extensively studied barrier function in 3 D models. The table also seems to indicate that the current manuscript is not so strongly novel as the authors would suggest.

Results
The quality of the epidermis is overstated and not better than 3D static models which are commercially available (MatTek, EpiSkin, CellSystems) or the multitude of inhouse models. Indeed  Fig 3 images show extremely poor epidermal stratification and differentiation in the chip with no clear basal layer, stratum spinosum, granular layer or stratum corneum. The layers are disorganized as shown by no localized loricrin (straum granulosum only), K10 (all suprabasal cells only) or K14 (basal layer only) expression as is repeatedly shown in 3D static models and skin. This indicates that contrary to what the authors write, superior epidermis and barrier function is not achieved compared to many static 3D models. The claim about K14 basal, K10 suprabasal and loricrin granular expression is overstated. Of note, loricrin is never expressed in basal keratinocytes so it is not surprising that the planar-liquid model does not express it. Thicker stratification is caused by air liquid exposure compared to wet / submerged exposure in 3D models. This then correlates to being less leaky and improved barrier. Of note, the condition of the planar/liquid cannot be used to represent current state of the art 3D static models as it is probably very wet (not air-exposed) due to leaking of medium through the hydrogel. This again questions how good this "novel" epidermal model really is.
Tilting of the chip only enabled the epidermis to remain dry as in standard 3D epidermal models, this in turn stimulates differentiation and improved barrier properties. This has been described by many others using the transwell system. Page 6 line 148: you do not show that enhanced ERK activation increases proliferation only that ERK expression in increased. This claim is overstated. The discussion is well writen and the Methods section is clear to follow.

General)
Before submitting our revised version of the manuscript, we would like to express our gratitude for the reviewers' insightful suggestions and insightful comments, which helped us to further improve our manuscript by elaborating our findings and statements.

Specific)
Reviewer-1 I believe that the majority of the points raised regarding the development of the model and the additional experiments have been adequately addressed by the authors.
The manuscript has significantly improved, however, there remain issues that must be addressed.

(Title) I would suggest rephrasing the revised title as it is very confusing in its current form. For instance, it is
not clear what "the innervated epidermis" refers to and what the model is.
We deeply appreciate the reviewer's comments. We revised the abstract as follows.
We fully understand and agree with the reviewer's comments, and revised the manuscript as follows.
(Revised manuscript) (Page 3, line 8) Free nerve endings of peptidergic or non-peptidergic C-fibers are mainly located close to keratinocytes in the spinous layer or granular layer of the epidermis, providing the structural basis for functional interaction such as synaptic-like contacts [9][10][11][12] .
(Page 3, line 18) However, the traditional 2D coculture systems have failed to spatially locate a cell or cell portion (e.g., the axon and cell body of a neuron) and to selectively analyze and probe specific cells. Cultured keratinocytes also suffer from morphological and functional limitations [15,[17][18]21] . The keratinocytes in vivo are existed in proliferating states at the basal layer of epidermis, and they undergo differentiation to form spinous, granular and cornified layer (Fig. 1a) [22] . 3D insert culture wells and microfluidic chips have been developed and further technologically improved by designing 3D culture conditions for epidermal morphogenesis and cell-customized compartmentalization for co-culture [13,[19][20][23][24][25][26] .
6. (Introduction and Results) Note that sensory neurons are pseudounipolar neurons and do not have dendrites.
The reference to dendrites is confusing. (Response) We appreciated your helpful comment. We revised the manuscript as follows (Page 5, line 2).

(Revised manuscript)
Keratinocytes loaded into the epidermal channel grow on one side of extracellular matrix (ECM) hydrogel and interact only with axons but not with neuronal soma, enabling localized axon-keratinocyte interaction studies like in vivo physiology (Fig. 1a and c).

(Introduction and Results)
All references throughout the manuscript to "sensory nerves" must be replaced with the appropriate terminology. In culture, it would generally be sensory axons or neurites. (Response) We deeply appreciate the reviewer's comments. We revised the manuscript as follows ( We appreciated the reviewer's helpful comments. We revised the manuscript as follows (Page 9, line 13).

(Revised manuscript)
Increased number of CGRP+ TRPV1+ fibers and TRPV1+ fibers from co-cultured sensory neurons could also be the reason for the increased CGRP. We fully understand and agree with the reviewer's comment. We revised the figure 1a as follows.

Reviewer: 2
The manuscript has been extensively revised and improved English needs editing. In the results section the quality of the epidermis is overstated and needs to be down-played a bit. Having said this, it is exceptional to be able to do so many readouts and to image whilst the culture is in the chip thus enabling cell cell interactions to be investigated without disrupting the tissue by having to remove it the chip first. The neuronal-epidermal interactions are very impressive. (Response) Thank you for your feedback. The English editor carefully edited the revised manuscript. We've attached the editor's proof.
Carefully, an exaggeration regarding the quality of the epidermis was revised. The response will be added to the second question posed by the reviewer.

(Introduction) supplimentary table 1 is unclear as not all abbreviations and colours are explained.
Importantly it is incomplete as many 3D skin models show e.g. loricrin (in stratum granulosum) and many authors have extensively studied barrier function in 3 D models.
The table also seems to indicate that the current manuscript is not so strongly novel as the authors would suggest. (Response) We greatly value the reviewer's insightful comments. Among the numerous in vitro skin models, we focused on the models that contained sensory neurons. As a result of the reviewer's suggestions, we conducted additional research, added Table 1 to the supplementary materials, and revised the manuscript accordingly (Page 3, Introduction).
As the reviewer noted, barrier function has also been investigated in numerous 3D skin models including dermis mimicking layer with fibroblasts or endothelial cells, but not in 3D in vitro skin models containing sensory neurons. However, the traditional 2D coculture systems have failed to spatially locate a cell or cell portion (e.g., the axon and cell body of a neuron) and to selectively analyze and probe specific cells. Cultured keratinocytes also suffer from morphological and functional limitations [15,[17][18]21] . The keratinocytes in vivo are existed in proliferating states at the basal layer of epidermis, and they undergo differentiation to form spinous, granular and cornified layer (Fig. 1a) We value the reviewer's feedback. We concur with the reviewer that the developed skin layer structure in the microfluidic device is inferior to that of commercially available models. Please note, however, that the To model the sensory innervation to epidermis [11] , we first adapted the slope-ALI method to induce the epidermal differentiation (Fig. 1b) [31,[38][39]43] . Enhanced ERK activation of keratinocytes was observed at 30 min after slope-ALI culture, and 3 days of slope-ALI culture helped the keratinocytes to proliferate and finally to form thicker epidermal-like layers in histological (Fig. 3a-c, 3l-m, and Supplementary Fig. 5). Tilting angle of the chip after the exposure of keratinocytes to the ALI reduced the hydrostatic pressure to the keratinocyte layer to help the differentiated keratinocytes make better alignment ( Fig. 1b and Supplementary Fig. 4). The epidermal-like layer expressed the markers of basal (cytokeratin 14 + , K 14), suprabasal (cytokeratin 10 + , K 10), and granular (loricrin + , for late-stage differentiation) layers. Keratinocytes cultured under planar-liquid condition mainly has basal keratinocytes without loricrin + cells (Fig. 3d-h). … 3. (Results) Thicker stratification is caused by air liquid exposure compared to wet / submerged exposure in 3D models. This then correlates to being less leaky and improved barrier. Of note, the condition of the planar/liquid cannot be used to represent current state of the art 3D static models as it is probably very wet (not air-exposed) due to leaking of medium through the hydrogel. This again questions how good this "novel" epidermal model really is.

(Results)
Tilting of the chip only enabled the epidermis to remain dry as in standard 3D epidermal models, this in turn stimulates differentiation and improved barrier properties. This has been described by many others using the transwell system.

(Response)
The reviewer's remarks are certainly accurate. We utilized the air-liquid interface culture condition, which is already known to facilitate epidermal differentiation.
The first accomplishment of this research is the confirmation that a differentiated epidermal layer with barrier function can be reconstituted in a microfluidic channel. It permits real-time imaging, sample volume reduction, spatial patterning or compartmentalization of multiple cell types and hydrogels, and precise regulation of cell-cell communication and interaction in in vitro skin research. As the reviewer noted, we did not observe a fully differentiated epidermal layer in our model; however, we were able to evaluate the sensory innervation to the epidermal-like layer using a variety of histological and functional assays. As the second achievement, it may be sufficient to state that peptidergic sensory innervation was induced in the epidermal-like layer containing K10+ We appreciated the reviewer's comment. Thank you.

Reviewers' Comments:
Reviewer #1: Remarks to the Author: The manuscript by Ahn et al., describes the reconstitution of an epidermal layer, with the accompanying innervation by the sensory neurons, in a microfluidic cell culture platform. The authors further provide proof of concept for the versatility of this microfluidic platform for disease modelling and studying the interactions between the sensory neurons and keratinocytes. The manuscript makes a significant contribution to the tissue-on-the-chip approaches to in vitro disease modelling.
The authors have thoroughly addressed all my questions and comments and I recommend the publication of the manuscript. The authors answer in the rebuttal is not true "Please note, however, that the commercially available models have only epidermis." See e.g. MatTeks EpiDerm FT and Phenion FT skin models, in addition to many research grade models ""Technically, a slope-air liquid interface (slope-ALI) culture was applied to provide an air contact necessary for epidermal differentiation, demonstrating advancements in keratinocyte development in terms of epidermal morphogenesis, differentiation, and barrier function."" This statement made by the authors is incorrect as indicated previously. This statement implies improvements in general, but this model has very inferior epidermal differentiation compared to static models in general. So what is it comparing to?
Keratinocytes cultured under planar-liquid condition mainly has basal keratinocytes without loricrin+ cells (Fig. 3d-h)." This text is not correct and is misleading. The K14, K10 and loricrin are intermitently expressed throughout the basal and suprabasal layers and not restricted to their correct skin locations. This indicates that stratification ocurs but abnormal differentiation in their culture.
Text description of fig 4J is also incorect. In the upper panel it appears that K14 (basal) may be above K10 (suprabasal which is opposite to lower image . The images do not show detailed enough histology to make any claims on epidermal quality or histology. This is not a superior epidermal culture as so emphasized throughout the manuscript.
Discussion: "forming highly organized epidermal-like layer co-influencing with innervated sensory neurons." again is over stated regarding the epidermis

Responses to Reviewer 1
The Thank you so much for your thoughtful comments. We deeply appreciate your valuable time for review procedure.
Responses to Reviewer 2 Q1) General: The manuscript is interesting but the novelty is still not clear compared to references in suppl table 1 and the epidermis quality is still extremely overstated. (Answer) We would like to begin by expressing our gratitude for reviewer 2's insightful suggestions and comments, which assisted us in refining our manuscript's findings and statements.
In this revision, -We elaborated further on the three key points of reviewer 2's persistent questions regarding our paper: epidermal quality, novelty, and value of coculture system.
-We have also revised the description of the manuscript to make it more clear, including the title, abstract, introduction, results, and discussions, by actively considering the reviewer's comments (marked in blue in the manuscript). The revised parts will be displayed in response to question 2. In the previous revision, we clarified the distinction between our model and previous models by revising the abstract, introduction, and discussion to reflect our understanding and agreement with the reviewer's point. In this revision, we attempted to clarify the originality of our model in the abstract, introduction, and discussion.
The comparison between our model and the references on which the reviewer commented is provided below.

supplementary ref 20; [Acta Biomater. 82, 93-101 (2018)]
* co-culture modeling; -The iPSC-derived sensory neurons (or mouse DRG neurons) and human keratinocytes are sequentially incorporated into the 3D collagen sponge, like insert culture system. And sponges were lifted up at air-liquid interface to promote keratinocyte differentiation for 17 days. ⇒ our system was differentiated for 5 days.
* Structural reconstitution; -They assessed for sensory neurons by immunofluorescent staining of the markers BRN3A, β3Tub, SP, NFM, TrkA and TRPA1 along with Ramp1 (a CGRP receptor) in sensory neurononly cultured groups, but not the cocultured group. ⇒ composition and structural patterning of sensory neuronal subsets were not quantified. They failed to promote axonal outgrowth from the neuronal cell layer into the epidermal layer. There was no evidence of an anatomically innervated epidermal layer in their model. -They quantified SP and CGRP release from neurons in fibroblasts embedded collagen sponge, without epidermis by ELISA ⇒ The authors provided no obvious evidence for the transmission property of co-cultured neurons and keratinocytes. They did not directly verify the functional integrity and subtype-specific integration of the nerve cells and keratinocyte layer. * Application of model; -The authors described in the discussion 'We intend to develop disease-specific skin models and analyze the potential effect of sensory nerves in these models after induction of neuropeptide release upon stimulation with TRPV1 agonists. Such a model would be particularly well-suited for the modeling of psoriasis, since this disease is highly suspected to be modulated by skin innervation. It represents an optimal base to build an immunocompetent skin model, with a strong potential for personalized medicine approaches.', but provided no obvious evidence for their descriptions.

ref 20; [Biomaterials 113, 217-229 (2017)]
* co-culture modeling; -They used a transwell insert to create a vertically stacked epidermal layer from keratinocytes on a hydrogel-based dermal layer containing fibroblasts. -The authors displayed K10 and filaggrin expression in the monocultured keratinocyte layer and they validated epidermal layer development with two-photon reconstruction of the cross section of innervated HSE ⇒ cellular and structural development of the epidermal layer was not assessed. They have not verified the functional integrity (barrier function) of the epidermal layer. There was no evidence of the quality of the epidermal layer in their model.
-They assessed for sensory neurons by immunofluorescent staining of the marker NFM in the cocultured group. ⇒ composition and structural patterning of sensory neuronal subsets were not quantified. They failed to promote axonal outgrowth from the neuronal cell layer into the epidermal layer. There was no evidence of an anatomically innervated epidermal layer in their model. -They analyzed calcium imaging on innervating HSE after the capsaicin addition without quantification of SP or CGRP release from neurons. ⇒ The authors provided no obvious evidence for the transmission property of co-cultured neurons and keratinocytes. They did not directly verify the functional integrity and subtype-specific integration of the nerve cells and keratinocyte layer. 3. Our co-culture system * co-culture modeling; -We presented a 3D microfluidic model for coculture and analyzes 3D interactions of keratinocytes and sensory neurons in vitro. Technically, a slope-air liquid interface (slope-ALI) culture was applied to provide an air contact necessary for epidermal differentiation without additional insert devices, demonstrating advancements in keratinocyte development in terms of epidermal differentiation, cell layering, and barrier function compared to conventional microfluidic chip systems using planar liquid culture. It was also shown that the hydrogelbased multi-channel system recapitulated the cellular/subcellular compartmentalization and cell-cell/cell-matrix interactions, leading to the physiologically relevant organization of the innervated epidermal-like layer and enabling functional analysis in a cell-type-specific manner, such as the in-situ permeability assay of the epidermis and sensory transmission assay initiated by topical stimulation to epidermal keratinocytes. * Structural reconstitution; -Although not as perfect as the transwell insert system, our slope-ALI culture and coculture system generated a more stratified epidermal layer-like structure than the planar-liquid culture and ref 20, showing more intense, continuous distribution, and cellular layering K10+ suprabasal-like keratinocytes over the K14+ basal-like cells. We demonstrated the structural and functional quality of epidermal layers with various experimental methods.
-Although IB4 + neurons failed to recapitulate the complete anatomy of the native skin (not innervate into the deep epidermis), we recapitulated cellular and histological structures of the innervated epidermis more successfully than conventional 3D transwell insert culture or microfluidic culture system methods (Fig 3-5).
* Functional integrity of sensory neurons and keratinocyte epidermal layer; -Cellular contacts and functional interaction between keratinocytes and neurons were observed in our innervated epidermal-like layers (Fig 5).
-We confirmed the nociceptive transduction by single-cell calcium imaging and SP/CGRP release quantification to evaluate the response of sensory neurons after topical treatment of (capsaicin, 4-PDD) on the epidermis (Fig. 3-5). * Application of model; -We modeled epidermal keratinocyte-sensory neuron crosstalk in our platform under hyperglycemic conditions to replicate hyperglycemia-induced diabetic neuropathy by simulating structural and functional changes in normal and disease states (Fig. 6) and demonstrated its feasibility as a model for investigating the underlying mechanism of the pathological condition.

⇒ In conclusion;
-Compared to the quality of the reconstituted epidermis layer, the 3D innervated skin models based on the conventional transwell system demonstrated insufficient sensory innervation data, which is considered a fundamental limitation of the transwell system from an analytical standpoint. We believed that the following features of the microfluidic system would enable it to overcome the aforementioned limitations: (transparency of materials, horizontally arranged channels, micro-scale culture chamber), by slope-ALI culture, optimized ECM hydrogels, and media in compartmented channels. We simulated 3D innervated epidermal layer on a microfluidic chip, and investigated how keratinocytes forming epidermal-like layer and innervating sensory neurons influence each other, and innervated epidermal layer. After confirming the structural and functional integration of, disease modeling was feasible.
-Our system conceptually aimed to improve and recapitulate the innervated epidermal model structurally and functionally on the microfluidic chip, not the insert-based system. To overcome limitations of 2D or 3D coculture systems, our system enables histological recapitulation, such as subcellular compartmentalization of neurons and FNEs in epidermal layers, and functional analysis in a cell type-dependent manner or in situ functional assays such as ca2+ transient and epidermal barrier testing. Moreover, we advanced the epidermal development on the chip by the slope-based culture to recapitulate the ALI culture, which was only possible in a 3D insert culture system.
Based on the comparison, we revised the manuscript as follows; (revised manuscript ; marked blue in pages [3][4] However, the traditional 2D coculture systems have failed to spatially locate a cell or cell portion (e.g., the axon and cell body of a neuron) and to selectively analyze and probe specific cells. Cultured keratinocytes also suffer from morphological and functional limitations [15,[17][18]21] . The keratinocytes in vivo have existed in proliferating states at the basal layer of the epidermis, and they undergo differentiation to form a spinous, granular, and cornified layer (Fig. 1a) [22] . 3D transwell culture platforms and microfluidic chips have been developed and further technologically improved by designing 3D culture conditions for epidermal morphogenesis and cell-customized compartmentalization for co-culture [13,[19][20][23][24][25][26] . In the 3D transwell insert culture system, a full-thickness human skin model with histological and functional properties that exhibit physiological similarity to in vivo skin was developed, but a reliable innervated skin model has yet to be reported [20,[23][24][25][27][28][29][30][31] . A recently reported spongebased co-culture model, like the transwell insert culture, also failed to mimic the anatomical distribution of intraepidermal free nerve ending and axon patterning, notwithstanding the well-differentiated epidermal layer (Supplementary Table 1) [20] . The advantages of microfluidic chips, commonly referred to as lab-on-a-chip (LoC) or cell chips [19,34] , have made them attractive candidates to replace traditional experiments, by reducing the sample volume and the cost of reagents, and providing investigators with substantially precise control and predictability of the spatiotemporal dynamics of the cell microenvironments and fluids [19,34] . In particular, the advantages of the spatiotemporal control allow researchers to closely recapitulate in vivo functions (both normal and disease states) by integrating several well-understood components into a single in vitro chip. However, reliable skin-nerve interactions and communication in the anatomically innervated epidermis have not yet taken advantage of microfluidics because they are based on the structure of vertically stacked systems, such as transwell insert cultures [16,19,[35][36] .
This work presents a new microfluidic model for coculture and analyzes 3D interactions of keratinocytes and sensory neurons in vitro. Technically, a slope-air liquid interface (slope-ALI) culture was applied to provide an air contact necessary for epidermal differentiation without additional devices, demonstrating advancements in keratinocyte development in terms of epidermal differentiation, cell layering, and barrier function compared to conventional microfluidic chip systems using planar liquid culture. It was also shown that the hydrogel-based multi-channel system recapitulated the cellular/subcellular compartmentalization and cell-cell/cell-matrix interactions, leading to the physiologically relevant organization of the innervated epidermal-like layer and enabling functional analysis in a cell-type-specific manner, such as the in-situ permeability assay of the epidermis and sensory transmission assay initiated by topical stimulation to epidermal keratinocytes. Finally, we modeled epidermal keratinocyte-sensory neuron crosstalk in our platform under hyperglycemic conditions mimicking acute diabetes and demonstrated its feasibility as a model for investigating the underlying mechanisms of the pathological condition.
(revised manuscript ; marked blue in pages 5) This cellular compartmentalization allows two independent cells to be conducted on a single device maintaining cellular identity and function, and also allows to selectively analyze and/or probe specific cells and cell portions (e.g., the axon and cell body in a neuron) that cannot be done in 2D and transwell insert co-culture system (Fig.  1c). Each axon-guiding microchannel is individually filled by physiologically relevant ECM hydrogel, i.e., type 1 collagen, acting as a layer of acellular dermal ECM, yet exclusively without fibroblasts [22,33,37] . After seeding DRG neuron cells (in the soma channel) and human epidermal keratinocytes (HEK, in the epidermal channel) sequentially, the medium in the keratinocyte channels was emptied and the cell-filled chip was tilted to maintain above 30 degrees tilt to mimic the air-liquid interface (slope-ALI culture), a common and critical microenvironment for the skin cell differentiation (Fig. 1b, 1d, and Supplementary Fig. 1 and 4a-c) [31,[38][39] . The developed microfluidic chip enables various imaging, biochemical and functional analyses such as axonal response testing and integrity/permeability tests, which can be conducted directly on the innervated epidermison-chips, thus improving the limitations of conventional transwell insert culture or previous microfluidic culture systems (Fig. 1c).
(revised manuscript ; marked blue in pages 6-7) To model the sensory innervation to the epidermis [11] , we first adapted the slope-ALI method to induce epidermal differentiation (Fig. 1b) [31,[38][39]43] . Our slope-ALI method rapidly initiates ERK activation and the proliferation of keratinocytes than the planar-liquid method, resulting in thicker epidermal-like layers (Fig. 3a-c, 3k-l, and Supplementary Fig. 6). This method developed multicellular epidermal differentiation such as the basal (cytokeratin 14 + , K 14), suprabasal (cytokeratin 10 + , K 10), and granular (loricrin + , for late-stage differentiation) cells compared to the planar-liquid method: which consists mainly of K14 + keratinocytes but few K10 + and loricrin + cells (Fig. 3d-f). The K14 + and K10 + keratinocytes of the slope-ALI method formed the suprabasal layer just above the basal layer like human epidermal tissue, showing a structurally more organized cell layer than the planar-liquid method (Fig. 3g). Under slope-ALI conditions, undulating micropatterned structures were noticed in the keratinocytes layer, like Rete ridge (RR) in natural human skin which has never been noticed in current tissue-engineered or 3D skin equivalents ( Supplementary Fig. 7) [42] . The keratinocyte layer in slope-ALI condition was tortuous but tightly interconnected showing a strong barrier function to 3.984 kDa FITC-conjugated dextran, consistent with more intense and continuous distribution results (Fig. 3g). It also showed enhanced blocking for the diffusive transport from the epidermal channel to the soma channel, consequently facilitating cell-type-specific functional analysis (Fig. 1c). Taken together, these results indicate that our slope-ALI culture can accelerate the proliferation of keratinocytes and their aligned layering during differentiation, reconstituting the tortuous layered epidermal keratinocyte layer.
(revised manuscript ; marked blue in pages 8) In the experiments, we found that NF200 + A-fibers (myelinated A-fibers) were more predominant than CGRP (peptidergic unmyelinated C-fibers) or IB4 (non-peptidergic unmyelinated C-fibers) positive neurons ( Fig. 4f and 4h-i). NF200 + A-fibers from co-cultured SNs have morphologically thinner and longer than those from monocultured SNs and usually terminate in dermal ECMs falling short of the epidermal layer ( Fig. 4f and 4h-i). CGRP + peptidergic neurons were significantly more in co-cultured SNs and were mainly confined in the region under the epidermal layer, some terminated within the epidermis as free nerve endings. Whereas IB4 + non-peptidergic fibers from mono-cultured SNs had more quantity in ECM hydrogel. Although IB4 + neurons from co-cultured SNs migrated through ECM hydrogel relatively longer than those from mono-cultured SNs, they did not innervate into the deep epidermis (granular layer) and failed to recapitulate the complete anatomy of the native skin ( Fig. 4f and 4h-i) [8][9][10] . Co-culture of SNs influence the development of epidermal keratinocytes in terms of morphogenesis and differentiation (Fig. 4j-k). When innervated, the epidermal-like layer grew on, not invading into the hydrogel, and presented enhanced alignment of K14, K10 and Loricrin ( Supplementary Fig.7a, 8a and 9a). In addition, the co-cultured epidermal keratinocyte layer showed a slight improvement in barrier function against 376.27 Da FITC-sodium (Fig. 4l-m). The co-culture of keratinocytes and sensory neurons in our slope-ALI microfluidic chip was proved to recapitulate cellular and histological structures of the innervated epidermis more successfully than conventional 3D transwell insert culture or microfluidic culture system.  Table 1